Holography was invented over sixty years ago by the physicist Dennis Gabor and is a technique that allows the light scattered from an object to be recorded and later reconstructed. Digital holography uses digital reconstruction of the diffraction patterns.
In digital holographic microscopy, a diffraction pattern, obtained by interference between a reference wave and an object wave that has interacted with an object of interest, may be detected and stored in a digital recording. By applying a reconstruction algorithm to the recorded diffraction pattern, an image or image signature of the object of interest may be obtained. Coherent or partially coherent light, collimated by a small aperture, may be used to illuminate an object in order to produce a diffraction pattern. This diffraction pattern may then be acquired by a high resolution optoelectronic sensor array. Such a lens-free holographic microscopy setup may produce a hologram of the object with phase information encoded in the diffraction images. Lens-free holographic imaging can provide an attractive low-cost solution for imaging small objects, such as, for example, microscopic objects (e.g., biological cells), because no expensive or complex optical components, such as high-quality optical lenses, are required.
Typical methods for holographic imaging in biological applications may be primarily based on in-line transmission geometry, in which coherent light from a light source illuminates a sample, which may be positioned on a glass substrate, and the diffraction or fringe pattern is recorded on an imager positioned on the opposite side of the sample with respect to the light source.
FIG. 1 illustrates an example typical holography setup for reconstructing a holographic image representing a transparent object. As shown, the setup 100 comprises a light source 102, an aperture 104, and a transparent surface 106, and an image sensor 108. The aperture 104 may be, for example, a pinhole. Other apertures are possible as well. The transparent surface 106 may be, for example, a glass substrate. Other transparent surfaces are possible as well.
As shown, the transparent surface 106 supports an object 110. The aperture 104 may collimate a light wave 112 emanating from the light source 102 to produce a substantially planar parallel coherent or partially coherent light wave near the object 110 after propagating unimpeded over a suitable distance between the aperture 104 and the object 110. The light wave may then interact with the object 110 (e.g., may undergo a phase shift due to changes in refractive index while passing through the object 110). A diffraction pattern formed by interference of an object wave component, which has interacted with the object 110, and a reference wave component, which has passed through the transparent surface 106 without interacting with the object 110, may then be recorded by the image sensor 108.
Seo et al. disclose a lens-free holographic cytometer. See Seo et al., Lab on a Chip, Vol. 9, Issue 6, pages 777-787 (2009). In particular, Seo et al. describe an imaging and reconstruction method and system that may allow for improved reconstructed images by providing rich texture information. The disclosed system may furthermore be used for characterization and counting of cells positioned on a complementary metal-oxide-semiconductor (CMOS) imaging chip. Seo et al. thus demonstrate that identification and/or characterization of a heterogeneous cell solution on a chip is feasible based on pattern recognition of the holographic diffraction pattern of each cell type.
However, holographic imaging using in-line transmission geometry may not be suitable for imaging non-transparent samples. Furthermore, dense or connected objects, such as biological tissue samples, may prevent the undistorted transmission of a suitable fraction of the wave through the sample in order to form a reference wave component. Therefore, when imaging such a non-transparent or dense sample, a suitable object wave component may desirably be obtained by reflection on the surface of the sample, instead of transmission through the sample.
When a high resolution is desired for small objects, the reflective-mode setup may become complicated. For example, FIG. 2 illustrates the working principles of a field portable reflection/transmission microscope based on lens-less holography, as described by Lee et al. See Lee et al., Biomedical Optics Express, Volume 2, Issue 9, pp. 2721-2730 (2011). As shown, the setup 200 is similar to that of a Michelson interferometer, and comprises a light source 202, an aperture 204, a beam-splitting device 206, a reflective surface 208, and an image sensor 210. The image sensor 210 may be, for example, a CMOS sensor chip. Other image sensors are possible as well.
The setup 200 may function as a lens-less reflection-mode microscope based on digital off-axis holography in which the beam-splitting device 206 and the reflective surface 208 are used to produce a tilted reference wave for producing an interference pattern by superposition on the reflected light from an object 212. Therefore, an off-axis hologram of the object 212 is created on the image sensor 210. The beam-splitting device 206 is an essential feature of the setup 200 for the interference of the reflected beam with the reflected light from an object to reconstruct the hologram.